1. Field of the Invention
This invention relates to an improvement in methods for detecting labeled molecules and especially biotinylated molecules. More particularly, the invention relates to an improvement in detection systems based on the interaction between biotin and either avidin or streptavidin.
2. Description of Related Art
The analysis and detection of minute quantities of substances in biological and nonbiological samples has become a routine practice in clinical, diagnostic and analytical laboratories. These detection techniques can be divided into two major classes: (1) those based on ligand-receptor interactions (e.g., immunoassay-based techniques), and (2) those based on nucleic acid hybridization (e.g., polynucleotide sequence-based techniques). Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody and an antigen complementary to it. Polynucleotide sequence-based detection techniques are characterized by a sequence of steps comprising the non-covalent binding of a labeled polynucleotide sequence or probe to a complementary sequence of the analyte under hybridization conditions in accordance with the Watson-Crick base pairing of adenine and thymine, and guanine and cytosine, and the detection of that hybridization.
In both classes of detection techniques, a nucleic acid probe or a polypeptide probe can be modified by a heterologous moiety and the heterologous moiety can be detected through a signaling moiety. The heterologous moiety contains at least two complex forming sites that form two different complexes, i.e., two kinds of complexes can be formed. The first complex forming site is utilized to attach the heterologous moiety to the probe (e.g., the polynucleotide or polypeptide) and the second (and additional) complex forming site(s) is(are) utilized to recognize (e.g., bind to) or activate the signaling moiety, with each complex formed being different and not interfering with each other. The signaling moiety contains a complex forming site that recognizes (e.g., binds to) the second complex forming site(s) of the heterologous moiety and a signaling site or portion that is capable of generating a signal directly or indirectly. The signal thus can be used to demonstrate the successful binding of the signaling moiety to the heterologous moiety.
An example of a nucleic acid probe or a polypeptide probe modified by a heterologous moiety is a biotinylated nucleic acid or a biotinylated polypeptide probe. Signaling moieties that are capable of creating a signal encompass a vast number of signal generating systems, including a moiety which generates a signal itself, e.g., a dye, a radioactive molecule, a chemiluminescent material, a fluorescent material or a phosphorescent material, or a moiety which upon further reaction or manipulation will give rise to a signal, e.g., a enzyme-linked system using an enzyme such as catalase, peroxidase, .beta.-glucuronidase, .beta.-D-glucosidase, .beta.-D-galactosidase, urease, glucose oxidase, galactose oxidase, or alkaline phosphatase. Substrates for such enzymes are well known and may produce a chromogenic, fluorescent or chemiluminescent signal for example. An example of a signaling moiety that has been utilized to detect a biotinylated probe is a modified avidin or streptavidin, normally modified by conjugation with an enzyme, such as horseradish peroxidase or alkaline phosphatase.
The identification and detection of biotinylated molecules attached to a target material in a sample immobilized on a solid phase support, such as a filter membrane, has generally been plagued by a background signal problem. The signaling moiety is localized on the solid support or solid phase and activated for reasons other than the presence of the target material. Nonspecific signal can occur under conditions wherein a labeled detector probe or a signaling moiety, such as a streptavidin-enzyme conjugate, indiscriminately binds for example (i) to the solid support or solid phase, such as a filter membrane, on which an assay is being conducted, or (ii) to a substance, usually a protein, used to block the solid phase. This localization of the labeled detector probe or signaling moiety for reasons other than the identification or recognition of a target material is often times referred to as non-specific binding. The consequence of such non-specific binding may be an inability to differentiate the desired signal from background noise.
Researchers have compensated for the variability in a solid phase membrane's propensity to bind the detector molecule or probe and/or the signaling moiety, e.g., enzyme-conjugated streptavidin, by treatments such as the use of heterologous DNA, an extended blocking step or a high salt washing step prior to addition of the indicator substrate (in this regard, see, R. K. Clark, et al., The Journal of Histochemistry and Cytochemistry, 34(11):1509-1515 (1986)). These steps have yielded various degrees of success, dependent to a large extent on the properties of the membrane itself, but improvements are still being sought. Nylon membranes, in particular, have continued to be a problem. Nylon membranes are used routinely by molecular biologists and have been the membrane of choice for nucleic acid work. Nucleic acids are readily immobilized on nylon membranes via UV irradiation. Nylon membranes also are very hardy and hold up well to multiple reprobing cycles involving high temperatures and chemical denaturants. Unfortunately nylon membranes generally exhibit considerable vendor-to-vendor, as well as lot-to-lot variability in the incidence of background signal problems, when used as a solid support, such as for nucleic acid hybridization assays, and particularly when used in conjunction with modified avidin and streptavidin as the signaling moiety.
It is therefore an object of this invention to provide a novel method of reducing background interference in ligand-receptor and nucleic acid hybridization-based assays and especially in such assays which rely on the biotin-avidin/streptavidin interaction for detection. The present invention is most particularly directed to nucleic acid hybridization assays which use biotinylated nucleic acid probes in connection with nylon solid phase supports.